Caveolae are well-known membrane domains found on the surface of most cells that are defined in part by a flask-shaped morphology, a filamentous coat on the cytoplasmic surface composed of caveolin and a high concentration of cholesterol relative to surrounding membrane. Caveolae engage in a distinctive endocytic activity capable of carrying molecules to the cytoplasm, to special endosomal compartments and to the ER. This domain is also critical for infection by pathogenic viruses and bacteria. Now that the caveolae pathway is better defined, important questions arise about the mechanism of invagination, budding and traffic, how caveolae achieve their unique protein and lipid composition, and the machinery involved in supplying structural lipids like cholesterol. This proposal outlines projects to address these three issues. The first project focuses on the mechanism of caveolae internalization. We have identified a new family of resident caveolae proteins that appear to regulate the internalization machinery by linking different isoforms of PKC to caveolin-1 and the internalization machinery. Experiments are planned to define how these caveolin adapters regulate internalization and identify the molecules involved. Caveolae contain a unique set of proteins that includes tyrosine kinase receptors and GPI anchored proteins. In aim 2, we will look at three potential mechanisms for controlling the localization of proteins to caveolae. One mechanism we will explore is based on recent findings that proteins surrounded by a lipid shell composed of cholesterol and sphingolipid are specifically retained by caveolae. Recently we found that PDGF stimulates the ubiquitination of PDGFRB in caveolae, so experiments are planned to determine the role of ubiquitination in controlling the exit of PDGFRB from caveolae. In addition, we will determine if retention of proteins in caveolae is controlled by extramembrane and intramembrane proteolysis. The third aim will focus on identifying the machinery that supplies cholesterol to caveolae and the mechanism cells use to sense the amount of cholesterol in caveolae and regulate delivery. These studies will focus on three proteins that have been implicated in maintaining caveolae cholesterol: caveolin, the cholesterol sensing molecule SR-BI and the cholesterol-regulated scaffolding protein OSBP. A better understanding of basic caveolae biology may lead to new strategies for treating human disease.